Described herein are high affinity nucleic acid ligands to vascular endothelial growth factor (VEGF). The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by EXponential enrichment
Neovascularization or angiogenesis is the process in which sprouting new blood vessels are formed from the existing endothelium in response to external stimuli that signal inadequate blood supply. Angiogenesis is generally rare under normal physiological conditions but frequently accompanies certain pathological conditions such as psoriasis, rheumatoid arthritis, hemangioma, and solid tumor growth and metastasis (Folkman and Klagsbrun (1987) Science 235:442-447; Kim et al. (1993) Nature 362:841-844). Several growth factors that are capable of inducing angiogenesis in vivo have been identified to date including acidic and basic fibroblast growth factors (aFGF, bFGF), transforming growth factors xcex1 and xcex2 (TGFxcex1, TGFxcex2), platelet derived growth factor (PDGF), angiogenin, platelet-derived endothelial cell growth factor (PD-ECGF), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF).
VEGF was originally purified from guinea pig ascites and tumor cell cultures as a factor that increases vascular permeability (Senger, D. R. et al. (1983) Science 219:983-985) and it has therefore also been referred to as vascular permeability factor (VPF). VEGF is a heat and acid-stable, disulfide-linked homodimer. Four isoforms have been described (121, 165, 189 and 206 amino acids, respectively) and are believed to be the result of alternative splicing of mRNA. Despite the presence of an identical N-terminal hydrophobic signal sequence in all molecular isoforms of VEGF, only the two lower molecular weight species are efficiently secreted (Ferrara, N. et al. (1991) J. Cell. Biochem. 47:211-218). The predominant VEGF isoform in most cells and tissues is the 165 amino acid species. Although VEGF is typically glycosylated, glycosylation is only required for efficient secretion but not for activity (Yeo, T-.K. et al. (1991) Biochem. Biophys. Res. Commun. 179:1568-1575; Peretz, D. et al. (1992) Biochem. Biophys. Res. Commun. 182:1340-1347). The amino acid sequence of VEGF is highly conserved across species and exhibits a modest but significant homology (18-20%) to PDGF A and B (Jakeman L. B. et al. (1992) J. Clin. Invest. 89:244-253; Ferrara et al. (1992) Endocrine Rev. 13:18-32).
Unlike other angiogenic growth factors, target cell specificity of VEGF is limited to vascular endothelial cells. The biological actions of VEGF are mediated through its interaction with specific cell-associated receptors which have been identified in the majority of tissues and organs (Jakeman, L. B. (1992) J. Clin. Invest. 89:244-253). Three high-affinity receptors for VEGF have been cloned to date: flt1, kdr/flk-1 and flt4 (Vaisman, N. et al. (1990) J. Biol. Chem. 265:19461-19466; de Vries, C. et al. (1992) Science 255:989-991; Galland, F. et al. (1993) Oncogene 8:1233-1240). These receptors belong to a family of transmembrane tyrosine kinases and bind VEGF with dissociation constants between 10xe2x88x9211 M to 10xe2x88x9212 M. Recent experiments have shown that binding of VEGF to its high-affinity receptors is significantly enhanced by heparin or cell surface-associated heparin-like molecules (Gitay-Goren, H. (1992) J. Biol. Chem. 267:6093-6098).
In addition to promoting the growth of vascular endothelial cells and inducing vascular leakage, VEGF is also known to induce the proteolytic enzymes interstitial collagenase, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) (Unemori E. et al. (1993) J. Cell. Physiology 153:557; Pepper, M. S. et al. (1992) Biochem. Biophys. Res. Commun. 189:824). These enzymes are known to play a prominent role in angiogenesis-related extracellular matrix degradation.
As a secreted and specific mitogen for endothelial cells, VEGF may be one of the major angiogenesis inducers in vivo. Several recent observations have supported this notion. For example, the expression of VEGF and its receptors accompanies angiogenesis associated with (i) embryonic development (Breier, G. et al. (1992) Development 114:521-532), (ii) hormonally-regulated reproductive cycle and (iii) tumor growth (Dvorak, H. F. (1991) J. Exp. Med. 174:1275-1278; Shweiki, D. et al. (1992) Nature 359:843-845; Plate, K. H. et al. (1992) Nature 359:845-848). It is relevant to note that aggressive tumor growth is accompanied by the generation of necrotic areas where oxygen and nutrient supplies are inadequate. Oxygen deprivation hypoxia) in tissues is a known angiogenesis stimulant. Interestingly, VEGF expression was found to be the highest in tumor cells facing the necrotic areas (Shweiki, D. et al. (1992) supra; Plate, K. H. et al. (1992) supra). It has therefore been suggested by these authors that VEGF plays a key role in hypoxia-induced angiogenesis.
Recent experiments with neutralizing monoclonal antibodies (MAbs) to VEGF have been especially meaningful for establishing that this growth factor is an important tumor angiogenesis inducer in vivo (Kim, K. J. et al. (1993) Nature 362:841-844). Immunocompromised (nude) mice injected with human rhabdomyosarcoma, glioblastoma or leiomyosarcoma cell lines rapidly develop tumors. Specific neutralizing MAb to VEGF were found to inhibit the growth of these tumors. The density of tumor vasculature was decreased in MAb-treated animals as compared to controls. The same MAb, on the other hand, had no effect on the growth rate of the tumor cells in vitro suggesting that the growth inhibition was not mediated at the cellular level and appears to be mediated by the 165-amino acid isoform of VEGF.
Herein described is the isolation and characterization of binding properties of a set of high-affinity nucleic acid ligands to VEGF. RNA, modified RNA, and ssDNA ligands are provided by the present invention. These ligands were selected from an initial pool of about 1014 RNA or DNA molecules randomized at thirty or forty contiguous positions. The evolved RNA ligands shown in FIGS. 2A-F bind VEGF with affinities in the low nanomolar range.
Also included herein are modified RNA ligands to VEGF. Such modified RNA ligands may be prepared after the identification of 2xe2x80x2-OH RNA ligands or by performing SELEX using a candidate mixture of modified RNAs. For example, 2xe2x80x2-NH2 pyrimidine RNA ligands to VEGF are described herein and the evolved ligands are shown in FIG. 9. Additionally post-SELEX modified RNA ligands are provided in Table 4.
Also included herein are ssDNA ligands to VEGF. The evolved ssDNA ligands are shown in Table 8.
The present invention includes the method of identifying nucleic acid ligands and ligand sequences to VEGF comprising:
a) contacting a candidate mixture of nucleic acids with VEGF, wherein nucleic acids having an increased affinity to VEGF relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;
b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and
c) amplifying the increased affinity nucleic acids, whereby nucleic acid ligands to VEGF may be identified.